Multiple-Drug Resistant Nasopharyngeal Streptococcus pneumoniae Isolated in Russia: Serotypes, Antimicrobial Susceptibility, and Molecular Characterization of the Emergent Serotype 13/ST2754 Lineage.

The pneumococcal population structure and drug resistance patterns are constantly changing worldwide. In this study, we described serotypes and antimicrobial susceptibility among 478 multiple-drug resistant (MDR) pediatric nasopharyngeal pneumococci recovered in 2010-2017. The majority of isolates (89.3%; n = 427) carried pneumococcal conjugate vaccine (PCV)13 serotypes, predominantly 6A/B, 14, 19A/F, and 23F. A non-PCV13 serotype capsule was detected in 44 (9.2%) MDR pneumococci, including serotypes 23A (n = 8), 13 (n = 7), 28F (n = 6), 11A (n = 5), and serogroup 35 (n = 10) isolates. The remaining seven (1.5%) MDR isolates were nontypeable. The majority of non-PCV13-serotype isolates were resistant to tetracycline, erythromycin, and clindamycin; most harbored both the ermB and mef genes. Among the 44 serotyped MDR non-PCV13 isolates, multilocus sequence typing analysis revealed 24 different sequence types (STs). ST2754 was the most abundant lineage demonstrating an unusual association with serotypes 13 (n = 7) and 9N (n = 1). The whole-genome sequencing-based analysis demonstrated that the serotype 13/ST2754 lineage was closely related to the serotype 13/ST2754 isolate recovered in Africa (Malawi) in 2013, possessed a Tn6002-like transposon carrying the erm(B) and tet(M) genes, and harbored additional virulence determinants, including arginine metabolism genes and a putative bacteriocin locus. Such a favorable genetic background may provide competitive advantages and potential for spreading and expansion of this clone among pneumococci. These data warrant further molecular monitoring of the genetic composition of the changing pneumococcal population.

Parallel detection of SARS-CoV-2 RNA and nucleocapsid antigen in nasopharyngeal specimens from a COVID-19 patient screening cohort.

OBJECTIVES: Reverse-transcription PCR (RT-PCR) is considered the most sensitive method for the detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). However, this method is relatively resource- and time-consuming. This study was performed to compare SARS-CoV-2 nucleocapsid antigen (N-Ag) testing using an enzyme-linked immunosorbent assay (ELISA) with SARS-CoV-2 RNA detection. METHODS: Parallel SARS-CoV-2 RT-PCR and quantitative N-Ag ELISA analysis was executed on nasopharyngeal specimens obtained during SARS-CoV-2 screening in a cohort of pre-hospitalization patients. RESULTS: In total, 277 specimens were examined, including 182 (65.7%) RT-PCR-positive specimens, which demonstrated a median cycle threshold (Ct) value of 27 (interquartile range (IQR) 23-35). The SARS-CoV-2 N-Ag was detected in 164 of the 182 RT-PCR-positive specimens (overall sensitivity 90.1%). Among the 95 RT-PCR-negative specimens, 72 were N-Ag-negative (specificity 75.8%). SARS-CoV-2 RT-PCR and N-Ag ELISA results demonstrated a strong agreement (Cramer's V = 0.668; P < 0.001). N-Ag concentrations spanned from 5.4 to 296 000 pg/ml (median 901 pg/ml, IQR 43-1407 pg/ml) and were inversely correlated with Ct values (Spearman's r = -0.720; P < 0.001). CONCLUSIONS: SARS-CoV-2 N-Ag ELISA results were in close agreement with RT-PCR results, and N-Ag concentrations were proportional to viral loads. Thus, SARS-CoV-2 quantitative antigen testing could be an additional diagnostic instrument for SARS-CoV-2.

A kinetic assay of total lipase activity for detecting lysosomal acid lipase deficiency (LAL-D) and the molecular characterization of 18 LAL-D patients from Russia.

Laboratory diagnostics of lysosomal acid lipase deficiency (LAL-D), a rare disorder associated with LIPA alterations, are based on the evaluation of LAL activity. In dry blood spots (DBS) submitted for LAL-D diagnostics (the screening cohort) over a two-year period or obtained from a cohort of retrospective LAL-D patients, we measured: (1) LAL activity using a two-reaction assay with 4-methylumbelliferone palmitate (4-MU-Palm) and Lalistat-2, a specific LAL inactivator; (2) total lipase (TL) activity by a 1-hour kinetic 4-MU-Palm cleavage reaction (no Lalistat-2). The TL activity was expressed as the area under the kinetic curve after 1 hour (TL-AUC1h) of the reaction and presented as the median (min-max). LAL activity was reduced in 30/537 individuals from the screening cohort, among which LIPA sequencing revealed six patients and one carrier. Overall, 16 (89%) individuals among six novel and 12 retrospective LAL-D patients carried at least one c.894G>A mutation (six were homozygous). The TL-AUC1h in nonLAL-D specimens with normal LAL activity (n = 90) was unambiguously higher (9471 [4015-23 585] RFU*h/punch) compared to LAL-D patients, including six new and nine retrospective patients (1810 [357-2608] RFU*h/punch). Importantly, in 13/15 examined nonLAL-D specimens with reduced LAL activity the TL-AUC1h was above a threshold of 2652 RFU*h/punch. Applying this threshold, the TL-AUC1h index discriminated all LAL-D patients (100% sensitivity) and 103/105 nonLAL-D specimens (98% specificity). Given that there is no need for Lalistat-2 and two parallel enzymatic reactions in conjunction with high sensitivity and specificity, the kinetic assay seems to be practical for LAL-D screening. SYNOPSIS: Lysosomal acid lipase deficiency responsible for Wolman disease and cholesterol ester storage disease could be reliably detected using a kinetic assay of total lipase activity with a fluorogenic substrate.

Changing serotype distribution and resistance patterns among pediatric nasopharyngeal pneumococci collected in Moscow, 2010-2017.

Serotype distribution and antimicrobial resistance were analyzed in 632 nasopharyngeal pneumococcal isolates collected at a single pediatric center in 2010-2017 before and following the introduction of the 13-valent pneumococcal conjugated vaccine (PCV13) in Russia in 2014. The mean prevalence of PCV13 serotypes was 77.7% in 2010-2015 with a significant decline to 58.5% in 2017, which was accompanied by an elevation in serotype 15B/C prevalence (15.1% in 2017), 66% and 26% of 15B/C-pneumococci related to ST1025 and ST1262, respectively. The rate of oxacillin, erythromycin, and clindamycin resistance has increased by 15-20 percentage points from 2010 to 2016, approaching a 40-45% prevalence in 2016. The resistance rates significantly increased over time only in a group of PCV13 serotypes. The growing resistance among serotype 14 pneumococci was associated with expansion of a multidrug-resistant clone of ST143. These results emphasize the need for close monitoring of the constantly changing pneumococcal population.